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Thermodynamic Characterization of Aminoglycoside-3•² University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2007 Thermodynamic Characterization of Aminoglycoside-3′- Phosphotransferase IIIa Can Özen University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Özen, Can, "Thermodynamic Characterization of Aminoglycoside-3′-Phosphotransferase IIIa. " PhD diss., University of Tennessee, 2007. https://trace.tennessee.edu/utk_graddiss/259 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Can Özen entitled "Thermodynamic Characterization of Aminoglycoside-3′-Phosphotransferase IIIa." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Engin H. Serpersu, Major Professor We have read this dissertation and recommend its acceptance: Michael Best, Hong Guo, Nitin Jain Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Can Özen entitled “Thermodynamic Characterization of Aminoglycoside-3′-Phosphotransferase IIIa.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. ______________________________ Engin H. Serpersu, Major Professor We have read this dissertation and recommend its acceptance: Michael Best______________________ Hong Guo_________________________ Nitin Jain_________________________ Accepted for the Council: ______________________________ Carolyn R. Hodges, Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Thermodynamic Characterization of Aminoglycoside-3′-Phosphotransferase IIIa A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Can Özen December 2007 Acknowledgements I want to thank Dr. Engin Serpersu for his guidance and support. I want to acknowledge my committee members, Drs. Nitin Jain, Hong Guo, and Michael Best for their helpful suggestions and input. I want to thank Drs. Elizabeth Howell, Elias Fernandez and Chris Dealwis for their valuable comments. I am grateful to all previous and present members of the Serpersu Lab who I’ve worked with for their help in my studies. I want to thank my wife Aysu Özen, my mother and father, Gökçen and Mehmet Özen, and my brother, Cem Özen for being there for me. Your love and support kept me going. ii Abstract Aminoglycoside-3′-Phosphotransferase IIIa is a widespread, promiscuous member of the phosphotransferase family of aminoglycoside modifying enzymes. This study provides results of combined calorimetry/NMR experiments to characterize and dissect the global thermodynamic properties of aminoglycoside–APH(3′)-IIIa complexes. Aminoglycoside binding to APH(3′)-IIIa is enthalpically driven with strong entropic penalty. 2′- and 6′-amino groups have significant contributions to the observed binding parameters. Formation of APH(3′)-IIIa complexes with substrate aminoglycosides shows a complex dependence on pH and is linked to protonation and deprotonation of both ligand and enzyme groups. We report pKa upshifts of ~1 unit for N2′ and N2′′′ groups of enzyme-bound neomycin B while the pKa of N6′ changes by 0.3 unit and N6′′′ experiences no shift. Isotopic solvent and heat capacity change studies strongly suggest differential effects and reorganization of solvent in kanamycin and neomycin class complexes of the enzyme. We also determined unusually high binding ΔCp values in the range of -0.7 to -3.8 kcal/mol·deg which were not explained by changes in the solvent accessible surface area. A break at 30°C was observed in the ΔCp plot and temperature- dependent backbone amide proton chemical shifts of four residues surrounding the binding site of kanamycin-APH(3′)-IIIa complex. These results may indicate specific solvent reorganization sites away from the binding site of the enzyme. iii Table of Contents Chapter Page PART I. INTRODUCTION ............................................................................................ 1 Aminoglycoside Antibiotics ........................................................................................... 2 Aminoglycoside Modifying Enzymes ............................................................................ 4 Aminoglycoside Phosphotransferase(3′)-IIIa ................................................................. 6 List of References ......................................................................................................... 10 PART II: THERMODYNAMICS OF AMINOGLYCOSIDE BINDING TO AMINOGLYCOSIDE-3′-PHOSPHOTRANSFERASE IIIA STUDIED BY ISOTHERMAL TITRATION CALORIMETRY ....................................................... 13 Abstract......................................................................................................................... 14 Materials and Methods.................................................................................................. 16 Results and Discussion ................................................................................................. 19 Conclusions................................................................................................................... 40 Acknowledgements....................................................................................................... 41 List of References ......................................................................................................... 42 PART III: DISSECTION OF AMINOGLYCOSIDE–ENZYME INTERACTIONS: A CALORIMETRIC AND NMR STUDY OF NEOMYCIN B BINDING TO THE AMINOGLYCOSIDE PHOSPHOTRANSFERASE(3′)-IIIA.................................... 45 Abstract......................................................................................................................... 46 Introduction................................................................................................................... 48 Materials and Methods.................................................................................................. 49 Results and Discussion ................................................................................................. 54 Conclusions................................................................................................................... 71 Acknowledgements....................................................................................................... 73 List of References ......................................................................................................... 74 PART IV: SOLVENT REORGANIZATION AND HEAT CAPACITY CHANGE IN AMINOGLYCOSIDE-APH COMPLEXES........................................................... 76 Introduction................................................................................................................... 77 Materials and Methods.................................................................................................. 79 Results........................................................................................................................... 81 Discussion..................................................................................................................... 89 Conclusions................................................................................................................. 100 Acknowledgements..................................................................................................... 101 List of References ....................................................................................................... 102 PART V. CONCLUSION............................................................................................. 107 APPENDIX.................................................................................................................... 110 iv VITA............................................................................................................................... 112 v List of Tables Table Page PART II Table 1. Thermodynamic Parameters for Aminoglycoside Binding to APH(3′)-IIIa (Binary Complex) at pH 7.5.............................................................................................. 20 Table 2. Protonation Coupled to Aminoglycoside Binding to APH(3′)-IIIa (Binary Complex) at pH 7.5........................................................................................................... 25 Table 3. Thermodynamic Parameters for Aminoglycoside Binding to APH(3′)-IIIa– CaATP (Ternary Complex) at pH 7.5............................................................................... 27 Table 4. Thermodynamic Parameters for Aminoglycoside Binding to APH(3′)-IIIa– CaATP (Ternary Complex) at pH 8.5..............................................................................
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